An extensive flow cytometric evaluation continues of patients with autoimmune lymphoproliferative syndrome (ALPS) and their extended family members, on the basis of characterization of the expanded double-negative T-cell and B-cell populations. Double-negative T-cells have been demonstrated to be alpha beta TcR, CD57+, HLA-DR+, and CD45RA+. This study has been extended to characterize the double-negative T-cells more completely including B220 expression and gamma-delta TcR T-cells in all ALPS patients. In addition, we have initiated expanded characterization of the B cells, directed at memory B cells using CD27 and B220 assessment in these patients. The observations in the B cells of ALPS patients are tied directly to an additional active protocol directed at the assessment of B220 expression on human lymphocytes. The relative deficiency in CD4/CD25 T cells that we have identified resulted in attempts at functional studies of immunoregulatory T cells but unanticipated problems developed and we were unable to demonstrate a consistent ex vivo indicator system for inhibition. Consequently, we changed our approach and have developed a quantitiative RTPCR assay for Fox P3 and have collected mRNA from T cells obtained from ALPS type Ia patients, family members and controls. These samples are now in the process of being evaluated for Fox P3 mRNA. In addition we have validated a flow cytometric assay for intracellular Fox P3 staining. This assay demonstrated that the bulk of Fox P3 expressing cells are CD4/CD25 T cells. We have begun an extensive evaluation of the Fox P3 expressing cells in control subjects and are in the process of preparing this data for publication. Our intial flow data from ALPS patients suggests that Fox P3 expression is normal despite the depression CD4/CD25 T cells but addional ALPS patients are required to validate this finding. Finally, we have also extended the Fox P3 PCR and flow based assays to ATL cells based on the possibility that this disease represents a leukemic expansion of immunoregulatory T cells. The ATL studies should be finalized in the next 6-8 months.